UBE2E3 (UbcH9) [untagged] E2 – Ubiquitin Conjugating Enzyme Alternate Names: UbcH9, UbcM2, Ubiquitin conjugating enzyme UbcH9 Cat. No. 62-0022-100 Quantity: 100 µg Lot. No. 1463 Storage: -70˚C FOR RESEARCH USE ONLY NOT FOR USE IN HUMANS CERTIFICATE OF ANALYSIS Page 1 of 2 Background Physical Characteristics The enzymes of the ubiquitylation Species: human Protein Sequence: pathway play a pivotal role in a num- GPLGSMSSDRQRSDDESPSTSSGSSDADQRD ber of cellular processes including Source: E. coli expression PAAPEPEEQEERKPSATQQKKNTKLSSKT regulated and targeted proteasomal TAKLSTSAKRIQKELAEITLDPPPNCSAGPK degradation of substrate proteins. Quantity: 100 μg GDNIYEWRSTILGPPGSVYEGGVFFLDITF SSDYPFKPPKVTFRTRIYHCNINSQGVI Three classes of enzymes are in- Concentration: 1 mg/ml CLDILKDNWSPALTISKVLLSICSLLTDCN volved in the process of ubiquityla- PADPLVGSIATQYLTNRAEHDRIARQWTKRYAT tion; activating enzymes (E1s), con- Formulation: 50 mM HEPES pH 7.5, jugating enzymes (E2s) and protein 150 mM sodium chloride, 2 mM The residues underlined remain after cleavage and removal ligases (E3s). UBE2E3 is a member dithiothreitol, 10% glycerol of the purification tag. of the E2 ubiquitin-conjugating en- UBE2E3 (regular text): Start bold italics (amino acid zyme family and cloning of the gene Molecular Weight: ~23 kDa residues 1-207) Accession number: NP_006348 was first described by Ito et al., 1999. UBE2E3 binds to the RING-finger Purity: >98% by InstantBlue™ SDS-PAGE proteins ARA54 and RNF8, thought to Stability/Storage: 12 months at -70˚C; act as E3 ligases in the ubiquitylation aliquot as required of nuclear proteins (Ito et al., 2001). The epithelial Na+ channel (ENaC) is regulated by UBE2E3 and the E3 Quality Assurance ligase NEDD4.2. UBE2E3 interacts with NEDD4.2 via its UBC domain Purity: Protein Identification: and ubiquitylation of ENaC occurs by 4-12% gradient SDS-PAGE Confirmed by mass spectrometry. InstantBlue™ staining NEDD4.2 binding the PY motifs of its Lane 1: MW markers E2-Ubiquitin Thioester Loading Assay: a, b and g subunits (Debonneville and Lane 2: 1 µg UBE2E3 The activity of UBE2E3 was validated by Staub. 2004). NEDD4.2 is a negative loading E1 UBE1 activated ubiquitin onto the regulator of ENaC and deletions in the active cysteine of the UBE2E3 E2 enzyme via PY motifs of the a and g subunits of a transthiolation reaction. Incubation of the ENaC cause Liddle’s syndrome, an in- UBE1 and UBE2E3 enzymes in the presence herited form of hypertension. The loss of ubiquitin and ATP at 30˚C was compared at of NEDD4.2 binding sites in mutated two time points, T0 and T10 minutes. Sensitiv- ENaC causes an increase in chan- ity of the ubiquitin/UBE2E3 thioester bond to the reducing agent DTT was confirmed. nel number at the cell surface and in- creased Na+ reabsorption by the dis- tal nephron, resulting in hypertension (Abriel et al., 1999). Continued on page 2 © Ubiquigent 2011. Unless otherwise noted, Ubiquigent, ORDERS / SALES SUPPORT UK HQ and TECHNICAL SUPPORT Ubiquigent logo and all other trademarks are the property of Ubiquigent, Ltd. International: +1-617-245-0003 International: +44 (0) 1382 381147 (9AM-5PM UTC) Limited Terms of Use: For research use only. Not for use in US Toll-Free: 1-888-4E1E2E3 (1-888-431-3233) US/Canada: +1-617-245-0020 (9AM-5PM UTC) humans or for diagnostics. Not for distribution or resale in Email: [email protected] Email: [email protected] any form, modification or derivative OR for use in providing services to a third party (e.g. screening or profiling) without the written permission of Ubiquigent, Ltd. www.ubiquigent.com Email [email protected] for enquiries regarding compound Dundee, Scotland, UK profiling and/or custom assay development services. Lot-specific COA version tracker: v1.0.0 UBE2E3 (UbcH9) [untagged] E2 – Ubiquitin Conjugating Enzyme Alternate Names: UbcH9, UbcM2, Ubiquitin conjugating enzyme UbcH9 Cat. No. 62-0022-100 Quantity: 100 µg Lot. No. 1463 Storage: -70˚C FOR RESEARCH USE ONLY NOT FOR USE IN HUMANS CERTIFICATE OF ANALYSIS Page 2 of 2 Background Continued from page 1 References: Abriel H, Loffing J, Rebhun JF, Pratt JH, Schild L, Horisberger JD, Rotin D, Staub O (1999) Defective regulation of the epithe- lial Na+ channel by Nedd4 in Liddle’s syndrome. J Clin Invest 103, 667-73. Debonneville C, Staub O (2004) Participation of the ubiquitin- conjugating enzyme UBE2E3 in Nedd4-2-dependent regulation of the epithelial Na+ channel. Mol Cell Biol 24, 2397-409. Ito K, Adachi S, Iwakami R, Yasuda H, Muto Y, Seki N, Okano Y (2001) N-Terminally extended human ubiquitin-conjugating en- zymes (E2s) mediate the ubiquitination of RING-finger proteins, ARA54 and RNF8. Eur J Biochem 268, 2725-32. Ito K, Kato S, Matsuda Y, Kimura M, Okano Y (1999) cDNA clon- ing, characterization, and chromosome mapping of UBE2E3 (alias UbcH9), encoding an N-terminally extended human ubiq- uitin-conjugating enzyme. Cytogenet Cell Genet 84, 99-104. © Ubiquigent 2011. Unless otherwise noted, Ubiquigent, ORDERS / SALES SUPPORT UK HQ and TECHNICAL SUPPORT Ubiquigent logo and all other trademarks are the property of Ubiquigent, Ltd. International: +1-617-245-0003 International: +44 (0) 1382 381147 (9AM-5PM UTC) Limited Terms of Use: For research use only. Not for use in US Toll-Free: 1-888-4E1E2E3 (1-888-431-3233) US/Canada: +1-617-245-0020 (9AM-5PM UTC) humans or for diagnostics. Not for distribution or resale in Email: [email protected] Email: [email protected] any form, modification or derivative OR for use in providing services to a third party (e.g. screening or profiling) without the written permission of Ubiquigent, Ltd. www.ubiquigent.com Email [email protected] for enquiries regarding compound Dundee, Scotland, UK profiling and/or custom assay development services. Lot-specific COA version tracker: v1.0.0.